Epstein–Barr virus (EBV) and human disease: facts,opinions and problems

The human herpesvirus, Epstein–Barr virus (EBV), has classically been associated with two pathologies with frequencies approaching 100%. One of these, Burkitt's lymphoma (BL), is of B-cell origin and the other, nasopharyngeal carcinoma (NPC), is a tumour of poorly differentiated epithelial cells. More recently, EBV has been identified with frequencies from a few percent to 100% (in one case) with a variety of other malignancies. These include Hodgkin's disease (HD; where in the west, the frequency of association is about 50%), sino-nasal T-cell lymphomas, lymphoepitheliomas, some sarcomas and breast cancers, other cancers from the head and neck, and lymphomas arising in patients with immune dysfunctions. Since EBV is ubiquitous, with the vast majority of the world's population having met and seroconverted to the virus, the diversity of tumours with which it has now been associated represents a substantial health burden. In a recent IARC monograph, EBV was classified as a group 1 carcinogen. Here, the data on BL and NPC, as they relate to geographical restrictions, viral strain variation, co-factors in disease, and genetic components are reexamined. We raise the question whether in their origins, these tumours genuinely reflect distinct and independent events, as deemed at present, or may represent a response by different cell types to common extracellular factors. For example, a situation in Kenya apparently existed in the past, where both BL and NPC were observed in ethnic Africans with roughly equal frequencies; more recently, in Kenya, EBV has been identified in nearly 100% of the tumours in children with HD. We also consider tumours where the viral association is reportedly of low frequency, and offer explanations for these data, including the possibility of loss of the viral genome once malignancy has been initiated. If this phenomenon occurs as a frequent secondary event, EBV could be an even greater health risk than presently believed.     

Clinical and immunological effect of intravesical interleukin-2 on superficial bladder cancer

TheBamHI Z EBV replication activator (ZEBRA) protein is involved in the switch from latency to productive cycle of Epstein-Barr virus. A recombinant ZEBRA protein was synthesized and assessed in enzymelinked immunosorbent assay (ELISA) for serum IgG response in nasopharyngeal carcinoma (NPC) patients. In 100 NPC serum samples that were positive for IgA to the EBV viral capsid antigen (VCA), 75% had IgG anti-ZEBRA antibodies. In contrast, only 3/83 (3.6%) serum samples from healthy donors and 2/50 (4%) from other cancers were positive for IgG to ZEBRA. Interestingly, in a selected group of 100 NPC sera negative for IgA to VCA, 25% contained IgG anti-ZEBRA antibodies. This suggests that the ELISA for IgG anti-ZEBRA may also identify earlier cases of NPC not detected by the conventional immunofluorescence test for IgA to VCA.     

Epstein-Barr virus infection: basis of malignancy and potential for therapy

The Epstein-Barr virus (EBV) is a human herpesvirus that is usually carried lifelong as an asymptomatic infection. EBV is the causative agent of infectious mononucleosis and has been linked to the development of several malignant tumours, including B-cell neoplasms such as Burkitt's lymphoma and Hodgkin's disease, certain forms of T-cell lymphoma, and some epithelial tumours, such as undifferentiated nasopharyngeal carcinoma and a proportion of gastric cancers. All these tumours are characterised by the presence of multiple extrachromosomal copies of the circular viral genome in the tumour cells and the expression of EBV-encoded latent genes, which appear to contribute to the malignant phenotype. An increasing understanding of the function of EBV latent genes and of the nature of the immune response to the virus is providing exciting new possibilities for the treatment of EBV-associated malignancies. For example, adoptive transfer of virus-specific cytotoxic T lymphocytes has already been of value in the treatment of EBV-positive B-cell lymphomas arising in post-transplant patients, and this approach is currently being investigated in other EBV-associated tumours. In addition, gene therapy offers the opportunity to deliver agents that might directly interfere with the function of specific EBV genes. This review summarises the role of EBV in malignancy. In particular, it focuses on the latent proteins as a basis for understanding how EBV might contribute to the process of transformation. Strategies to target EBV in tumours, potentially providing alternative therapeutic approaches, are also discussed.     

Resveratrol inhibits proliferation and survival of Epstein Barr virus-infected Burkitt's lymphoma cells depending on viral latency program

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenolic natural product, shows chemopreventive properties against several cancers, heart diseases, inflammation, and viral infections. Epstein Barr virus (EBV), a γ-herpesvirus, contributes to the development of several human cancers including Burkitt's lymphoma (BL). In this study, we asked whether treatment with resveratrol would affect the viability of EBV-positive BL cells displaying different forms of latency. We report here that resveratrol, regardless of EBV status, induces caspase-dependent apoptosis by arresting cell-cycle progression in G(1) phase. However, resveratrol strongly induced apoptosis in EBV(-) and latency I EBV(+) cells, whereas latency II and latency III EBV(+) BL cells showed a survival advantage that increased with the extent of the pattern of viral gene expression. Resveratrol-induced cell-cycle arrest and apoptosis occurred in association with induction of p38 MAPK phosphorylation and suppression of ERK1/2 signaling pathway. Moreover, NF-κB DNA-binding activity was inhibited in all BL lines except EBV(+) latency III cells. LMP1 oncogene, which is expressed in latency III phenotype, is involved with the higher resistance to the antiproliferative effect of resveratrol because siRNA-mediated inhibition of LMP1 greatly increased the sensitivity of latency III BL cells as well as that of lymphoblastoid cell lines to the polyphenol. We propose that a combined resveratrol/siRNA strategy may be a novel approach for the treatment of EBV-associated B-cell malignancies in which the viral pattern of gene expression has been defined.     

EB病毒DNA聚合酶基因的表达与纯化鼻咽癌病人诊断中的应用

以Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒（ＥＢＶ）ＤＮＡ聚合酶为抗原,建立了简便、快速、敏感和特异的鼻咽癌诊断方法。构建原表达载体ｐＲＳＥＴ－ＤＮＡ聚合酶及其亚克隆ＰＲＳＥＴ－Ａ１和ＢＬ２１（ＤＥ３）中表达的产物,经Ｗｅｓｔｅｒｎ－ｂｌｏｔ检测其抗原性并用于检测鼻咽癌（ｎａｓｏｐｈａｒｙｎｇｅａｌ ｃａｒｃｉｎｏｍａ,ＮＰＣ）病人血清中的抗体。在ＤＰＣ病人血清中存在抗ＥＢ病毒ＤＮＡ聚合酶的ＩｇＧ抗体,并证明     

Nasopharyngeal carcinomas and Epstein-Barr virus: from epidemiology and detection to therapy

Nasopharyngeal carcinomas (NPC) challenge clinicians and biologists in various fields including epidemiology, genetics, virology and immunology. These tumours have a striking geographical distribution. They are constantly associated with the Epstein-Barr virus (EBV) and contain a massive lymphocytic infiltrate. Their study has major implications especially at this moment while a pathological role of EBV is suspected in several other human epithelial malignancies (for example gastric, mammary and thyroid carcinomas). The North-South Workshop on Nasopharyngeal Carcinoma was held at the Institut Gustave-Roussy in early December 2003. Its main goal was to support the exchanges between clinical research on NPC in the South and basic research in the North. With regard to epidemiology and genetics, the main information was the possible existence of several susceptibility genes (including two of them on the 4p and 5p chromosomes). In virology, participants have emphasized the selection of peculiar EBV variants within the malignant cells and the expression of novel oncogenic viral proteins : LMP2 and BARF1. Cellular gene alterations also contribute to NPC development, especially inactivation of tumor suppressor genes located on the 3p chromosome. Therapeutic research was not forgotten. Hope of higher rate of cure relies on improved ballistic processes in radiotherapy (IMRT) and on the development of targeted therapeutics : induction of the lytic/productive viral cycle, gene therapy with conditional replicative adenoviruses, antitumor vaccination directed against the viral protein LMP2.     

Nasopharyngeal carcinoma: molecular pathogenesis and therapeutic developments

Nasopharyngeal carcinoma (NPC) is a prevalent tumour in southern China and southeast Asia, particularly in the Cantonese population, where its incidence has remained high for decades. Recent studies have demonstrated that the aetiology of NPC is complex, involving multiple factors including genetic susceptibility, infection with the Epstein-Barr virus (EBV) and exposure to chemical carcinogens. During development of the disease, viral infection and multiple somatic genetic and epigenetic changes synergistically disrupt normal cell function, thus contributing to NPC pathogenesis. NPC is highly radiosensitive and chemosensitive, but treatment of patients with locoregionally advanced disease remains problematic. New biomarkers for NPC, including EBV DNA copy number or methylation of multiple tumour suppressor genes, which can be detected in serum and nasopharyngeal brushings, have been developed for the molecular diagnosis of this tumour. Meanwhile, new therapeutic strategies such as intensity-modulated radiation therapy and immuno- and epigenetic therapies might lead to more specific and effective treatments.     

Direct sequencing and characterization of a clinical isolate of Epstein-Barr virus from nasopharyngeal carcinoma tissue by using next-generation sequencing technology

Epstein-Barr virus (EBV)-encoded molecules have been detected in the tumor tissues of several cancers, including nasopharyngeal carcinoma (NPC), suggesting that EBV plays an important role in tumorigenesis. However, the nature of EBV with respect to genome width in vivo and whether EBV undergoes clonal expansion in the tumor tissues are still poorly understood. In this study, next-generation sequencing (NGS) was used to sequence DNA extracted directly from the tumor tissue of a patient with NPC. Apart from the human sequences, a clinically isolated EBV genome 164.7 kb in size was successfully assembled and named GD2 (GenBank accession number HQ020558). Sequence and phylogenetic analyses showed that GD2 was closely related to GD1, a previously assembled variant derived from a patient with NPC. GD2 contains the most prevalent EBV variants reported in Cantonese patients with NPC, suggesting that it might be the prevalent strain in this population. Furthermore, GD2 could be grouped into a single subtype according to common classification criteria and contains only 6 heterozygous point mutations, suggesting the monoclonal expansion of GD2 in NPC. This study represents the first genome-wide analysis of a clinical isolate of EBV directly extracted from NPC tissue. Our study reveals that NGS allows the characterization of genome-wide variations of EBV in clinical tumors and provides evidence of monoclonal expansion of EBV in vivo. The pipeline could also be applied to the study of other pathogen-related malignancies. With additional NGS studies of NPC, it might be possible to uncover the potential causative EBV variant involved in NPC.     

Epstein-Barr virus-associated Burkitt lymphomagenesis selects for downregulation of the nuclear antigen EBNA2

Epstein-Barr virus (EBV) is etiologically linked to endemic Burkitt lymphoma (BL), but its contribution to lymphomagenesis, versus that of the chromosomal translocation leading to c-myc gene deregulation, remains unclear. The virus's growth-transforming (Latency III) program of gene expression is extinguished in tumor cells, and only a single viral protein, the EBV nuclear antigen (EBNA)1, is expressed via the alternative Latency I program. It is not known if BL arises from a B-cell subset in which EBV naturally adopts a Latency I infection or if a clone with limited antigen expression has been selected from an EBV-transformed Latency III progenitor pool. Here we identify a subset of BL tumors in which the Latency III-associated EBNA promoter Wp is active and most EBNAs are expressed, but where a gene deletion has specifically abrogated the expression of EBNA2. This implies that BL can be selected from a Latency III progenitor and that the principal selection pressure is for downregulation of the c-Myc antagonist EBNA2.     

An Epstein-Barr Virus Encoded Inhibitor of Colony Stimulating Factor-1 Signaling Is an Important Determinant for Acute and Persistent EBV Infection

Acute Epstein-Barr virus (EBV) infection is the most common cause of Infectious Mononucleosis. Nearly all adult humans harbor life-long, persistent EBV infection which can lead to development of cancers including Hodgkin Lymphoma, Burkitt Lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and lymphomas in immunosuppressed patients. BARF1 is an EBV replication-associated, secreted protein that blocks Colony Stimulating Factor 1 (CSF-1) signaling, an innate immunity pathway not targeted by any other virus species. To evaluate effects of BARF1 in acute and persistent infection, we mutated the BARF1 homologue in the EBV-related herpesvirus, or lymphocryptovirus (LCV), naturally infecting rhesus macaques to create a recombinant rhLCV incapable of blocking CSF-1 (ΔrhBARF1). Rhesus macaques orally challenged with ΔrhBARF1 had decreased viral load indicating that CSF-1 is important for acute virus infection. Surprisingly, ΔrhBARF1 was also associated with dramatically lower virus setpoints during persistent infection. Normal acute viral load and normal viral setpoints during persistent rhLCV infection could be restored by Simian/Human Immunodeficiency Virus-induced immunosuppression prior to oral inoculation with ΔrhBARF1 or infection of immunocompetent animals with a recombinant rhLCV where the rhBARF1 was repaired. These results indicate that BARF1 blockade of CSF-1 signaling is an important immune evasion strategy for efficient acute EBV infection and a significant determinant for virus setpoint during persistent EBV infection.     

Epstein-Barr virus nuclear antigen 1 replication and segregation functions in nasopharyngeal carcinoma cell lines

Nasopharyngeal carcinomas (NPC) are usually Epstein-Barr virus (EBV) positive, but, with the exception of C666-1 cells, these cells lose the EBV genomes when grown in culture. Maintenance of EBV requires the viral EBV nuclear antigen 1 (EBNA1) protein, which ensures the replication and mitotic segregation of the genomes through interactions with OriP. Here we compare the abilities of C666-1 and NPC cells that have lost EBV genomes to replicate and segregate OriP plasmids. We found that either cell line can replicate and maintain OriP plasmids for extended periods under conditions where low levels of EBNA1 are expressed but that high EBNA1 levels selectively interfered with mitotic segregation.     

Epstein-Barr virus (EBV)-negative B-lymphoma cell lines for clonal isolation and replication of EBV recombinants.

Previous experiments have demonstrated that positive selection markers recombined into the Epstein-Barr virus (EBV) genome enable the isolation of transforming or nontransforming mutant EBV recombinants in EBV-negative B-lymphoma (BL) cell lines (A. Marchini, J. I. Cohen, and E. Kieff, J. Virol. 66:3214-3219, 1992; F. Wang, A. Marchini, and E. Kieff, J. Virol. 65:1701-1709, 1991). However, virus has been recovered from a BL cell clone (BL41) infected with an EBV recombinant in only one instance (Wang et al., J. Virol. 65:1701-1709, 1991). We now compare the utility of four EBV-negative BL lines, BJAB, BL30, BL41, and Loukes, for isolating EBV recombinants and supporting their subsequent replication. Transforming or nontransforming EBV recombinants carrying a simian virus 40 promoter-hygromycin phosphotransferase (HYG) cassette were cloned by selecting newly infected BL cells for HYG expression. Most of the infected BL clones contained EBV episomes, and EBV gene expression was largely restricted to EBNA-1. Although the BJAB cell line was a particularly good host for isolating EBV recombinants (Marchini et al., J. Virol. 66:3214-3219, 1992), it was largely nonpermissive for virus replication, even in response to heterologous expression of the BZLF1 immediate-early transactivator. In contrast, approximately 50% of infected BL41, BL30, or Loukes cell clones responded to lytic cycle induction. Frequently, a substantial fraction of infected cells expressed the late lytic infection viral protein, gp350/220, and released infectious virus. Since BL cells do not depend on EBV for growth, transforming and nontransforming EBV recombinants were isolated and passaged.     

EBV编码miRNAs在病毒感染和肿瘤发生中的功能和意义

EB病毒(Epstein-Barr virus,EBV)是一种172 kb大小的线性双链DNA病毒,与鼻咽癌、淋巴瘤、胃癌等恶性肿瘤的发生密切相关. EBV编码的微小RNAs (miRNAs)可以调节病毒和宿主细胞基因的表达,并且在癌症发生发展中起着多种作用.本文综述了EBV编码的miRNAs (EBV-encoded miRNA,EBV miRNAs)在病毒感染和肿瘤发生、侵袭转移、抗凋亡、信号通路等方面的生物学功能,以及对于EBV相关肿瘤诊断标志物的潜在意义. EB病毒编码的miRNAs也可能成为进一步研究EBV相关肿瘤治疗的一个候选靶点.     

Genomic sequence analysis of Epstein-Barr virus strain GD1 from a nasopharyngeal carcinoma patient

To date, the only entire Epstein-Barr virus (EBV) genomic sequence available in the database is the prototype B95.8, which was derived from an individual with infectious mononucleosis. A causative link between EBV and nasopharyngeal carcinoma (NPC), a disease with a distinctly high incidence in southern China, has been widely investigated. However, no full-length analysis of any substrain of EBV from this area has been reported. In this study, we analyzed the entire genomic sequence of an EBV strain from a patient with NPC in Guangdong, China. This EBV strain was termed GD1 (Guangdong strain 1), and the full-length sequence of GD1 was submitted to the GenBank database. The assigned accession number is AY961628. The entire GD1 sequence is 171,656 bp in length, with 59.5% G+C content and 40.5% A+T content. We detected many sequence variations in GD1 compared to prototypical strain B95.8, including 43 deletion sites, 44 insertion sites, and 1,413 point mutations. Furthermore, we evaluated the frequency of some of these GD1 mutations in Cantonese NPC patients and found them to be highly prevalent. These findings suggest that GD1 is highly representative of the EBV strains isolated from NPC patients in Guangdong, China, an area with the highest incidence of NPC in the world. Furthermore, these findings provide the second full-length sequence analysis of any EBV strain as well as the first full-length sequence analysis of an NPC-derived EBV strain.     

Functional and physical interaction between p53 and BZLF1: implications for Epstein-Barr virus latency.

The p53 tumor suppressor protein, which is commonly mutated in human cancers, has been shown to interact directly with virally encoded from papillomavirus, adenovirus, and simian virus 40. The disruption of p53 function may be required for efficient replication of certain viruses and may also play a role in the development of virally induced malignancies. Infection with Epstein-Barr virus (EBV) has been associated with the development of B-cell lymphomas and nasopharyngeal carcinoma. Here we show that the EBV immediate-early protein, BZLF1 (Z), which is responsible for initiating the switch from latent to lytic infection, can interact directly in vitro and in vivo with the tumor suppressor protein, p53. This interaction requires the coiled-coil dimerization domain of the Z protein and the carboxy-terminal portion of p53. Overexpression of wild-type p53 inhibits the ability of Z to disrupt viral latency. Likewise, Z inhibits p53-dependent transactivation in lymphoid cells. The direct interaction between Z and p53 may play a role in regulating the switch from latent to lytic viral infection.